Investigation of association of genetic variant rs3918242 of matrix metalloproteinase-9 with hypertension, myocardial infarction and progression of ventricular dysfunction in Irish Caucasian patients with diabetes: a report from the STOP-HF follow-up programme.

BACKGROUND: Hypertension and/or myocardial infarction are common causes of heart failure in Type 2 diabetes. Progression to heart failure is usually preceded by ventricular dysfunction, linked to matrix metalloproteinase (MMP) mediated extracellular matrix changes. We hypothesise that the minor allele of genetic variant rs3918242 in the promoter region of the MMP-9 gene is associated with hypertension and/or myocardial infarction, with resultant progression of dysfunctional cardiac remodelling in patients with diabetes without symptomatic heart failure. METHODS: We genotyped 498 diabetes patients participating in the St Vincent's Screening TO Prevent Heart Failure (STOP-HF) follow-up programme for the rs3918242 single nucleotide polymorphism and investigated associations with the co-primary endpoints hypertension and/or myocardial infarction using a dominant model. We also evaluated resulting cardiometabolic phenotype and progression of ventricular dysfunction and cardiac structural abnormalities over a median follow-up period of 3.5 years. RESULTS: The CT/TT genotype comprised 28.1% of the cohort and was associated with a twofold higher risk of myocardial infarction (17.9% vs 8.4%), a reduction in ejection fraction and greater left ventricular systolic dysfunction progression [adjusted OR = 2.56 (1.09, 6.01), p = 0.026] over a median follow-up of 3.5 years [IQR 2.6, 4.9 years]. Conversely, rs3918242 was not associated with hypertension, blood pressure, pulse pressure or left ventricular mass index at baseline or over follow up. CONCLUSIONS: Diabetes patients with the minor T allele of rs3918242 in the STOP-HF follow up programme have greater risk of myocardial infarction, lower ejection fraction and greater progression of left ventricular systolic abnormalities, a precursor to heart failure. These data may support further work on MMP-9 as a biomarker of ventricular dysfunction and the investigation of MMP-9 inhibitors for heart failure prevention in diabetes, particularly in the post-infarction setting. ClinicalTrials.gov Identifier: NCT00921960.

Postprandial adiponectin and gelatinase response to a high-fat versus an isoenergetic low-fat meal in lean, healthy men.

OBJECTIVE: Evidence suggests that an acute systemic inflammatory response is invoked after consumption of a high-energy meal. Postprandial regulation of adiponectin, an adipose tissue-derived, anti-inflammatory hormone, and the gelatinases, matrix metalloproteinase (MMP)-2 and MMP-9, endopeptidases implicated in a diverse range of inflammatory processes, remain inconclusive. The aim of this study was to assess the postprandial effect of a high-energy (1212 kcal) meal on plasma adiponectin, MMP-2 and MMP-9 activity, glucose, insulin, triacylglycerols, total cholesterol, high-density lipoprotein cholesterol, and the differential effects on these parameters depending on whether the test meal was high fat (HF; 46 g fat, 1210 kcal) or isoenergetic and low fat (LF; 15 g fat, 1214 kcal energy). METHODS: Test meals were consumed by 17 lean, healthy men on two separate occasions with blood samples collected by venipuncture at baseline (0 h) and 1 and 3 h after consumption of each test meal. RESULTS: At baseline, no significant difference was seen in the parameters between the two groups, except for MMP-2, MMP-9, and total cholesterol. Over the 3-h postprandial period, no significant differential effect of the HF versus the LF test meal was observed on adiponectin, MMP-2, MMP-9, or on metabolic markers other than triacylglycerol, which increased significantly in response to the HF test meal (time × treatment, P = 0.002). When analyzed independent of time, MMP-2 (treatment, P = 0.006), MMP-9 (treatment, P = 0.022), and glucose (treatment, P = 0.026) were lower after consumption of the HF meal compared with the LF test meal. When analyzed independent of treatment, adiponectin increased over the 3-h postprandial period (time, P = 0.031), but there was no change in MMP-2 or MMP-9 (time, P = 0.503 and P = 0.525, respectively). Over the 3-h postprandial period, insulin (time, P < 0.001) and total cholesterol (time, P = 0.002) increased, whereas glucose (time, P < 0.001) and high-density lipoprotein cholesterol (time, P < 0.001) decreased. CONCLUSION: No differential effects of a HF versus a LF isoenergetic meal were seen on postprandial adiponectin or the gelatinases. Adiponectin increased in response to a high-energy meal independent of treatment, and the gelatinases were lower in response to the HF versus the LF isoenergetic meal, independent of time point. Given the considerable amount of time that humans spend in the postprandial state, additional research is necessary to further understand inflammatory changes in this state.

HIV-1 Tat clade-specific cytokine induction in monocytes/macrophages is not evidenced in total or Vγ9Vδ2 T lymphocytes.

HIV-1 Tat exhibits clade-specific cytokine induction in monocytes. We investigated if Tat clades A-D can alter tumour necrosis factor (TNF)-α and interferon (IFN)-γ production by total and Vγ9Vδ2 T cells in vitro. Tat clade B, but not C, augmented TNF-α production by THP-1 cells. However, Tat clades A-D did not affect TNF-α or IFN-γ production or secretion by resting or activated conventional and Vγ9Vδ2 T cells. Therefore, transactivation of cytokines by Tat is immune cell-specific.

Long-term statin therapy in patients with systolic heart failure and normal cholesterol: effects on elevated serum markers of collagen turnover, inflammation, and B-type natriuretic peptide.

BACKGROUND: The role of statin therapy in heart failure (HF) is unclear. The amino-terminal propeptide of procollagen type III (PIIINP) predicts outcome in HF, and yet there are conflicting reports of statin therapy effects on PIIINP. OBJECTIVES: This study determined whether there was an increase in serum markers of inflammation, fibrosis (including PIIINP), and B-type natriuretic peptide (BNP) in patients with systolic HF and normal total cholesterol and determined the effects of long-term treatment with atorvastatin on these markers. METHODS: Fifty-six white patients with systolic HF and normal cholesterol levels (age 72 [13] years; 68% male; body mass index 27.0 [7.3] kg/m(2); ejection fraction 35 [13]%; 46% with history of smoking) were randomly allocated to atorvastatin treatment for 6 months, titrated to 40 mg/d (A group) or not (C group). Age- and/or sex-matched subjects without HF (N group) were also recruited. Biomarkers were measured at baseline (all groups) and 6 months (A and C groups). RESULTS: Serum markers of collagen turnover, inflammation, and BNP were significantly elevated in HF patients compared with normal participants (all P < 0.05). There were correlations between these markers in HF patients but not in normal subjects. Atorvastatin treatment for 6 months caused a significant reduction in the following biomarkers compared with baseline: BNP, from median (interquartile range) 268 (190-441) pg/mL to 185 (144-344) pg/mL; high-sensitivity C-reactive protein (hs-CRP), from 5.26 (1.95 -9.29) mg/L to 3.70 (2.34-6.81) mg/L; and PIIINP, from 4.65 (1.86) to 4.09 (1.25) pg/mL (all P < 0.05 baseline vs 6 months). Between-group differences were significant for PIIINP only (P = 0.027). There was a positive interaction between atorvastatin effects and baseline hs-CRP and PIIINP (P < 0.01). CONCLUSIONS: Long-term statin therapy reduced PIIINP in this small, selected HF population with elevated baseline levels. Further evaluation of statin therapy in the management of HF patients with elevated PIIINP is warranted.

Ribavirin and interferon alter MMP-9 abundance in vitro and in HIV-HCV-coinfected patients.

BACKGROUND: Matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) are central to tissue remodelling during HIV-HCV infection. Here, we assess the potential for antiviral therapy to modulate MMP abundance in THP-1 monocyte/macrophages and LX-2 hepatic stellate cells, and in a coinfected patient cohort. METHODS: THP-1 and LX-2 cells were treated with ribavirin (RBV)/interferon-α (IFN-α) and select HIV antivirals. Venous blood was reserved from HIV-HCV-coinfected patients, HIV- and HCV-monoinfected patients, and healthy controls, with the HIV-HCV cohort being sampled again at day 3 and 14 subsequent to the start of combination therapy with RBV/pegylated IFN-α. Samples were subjected to gelatin zymography, real-time RT-PCR and/or ELISA, where appropriate. RESULTS: RBV/IFN-α decreased MMP-9 activity, and increased MMP-9 mRNA and protein expression in THP-1 cells, but not in LX-2 cells. Decreases in MMP-9 activity were mediated by IFN-α, which also attenuated RBV induction of MMP-9 activity and protein expression in THP-1 cells. Saquinavir and lopinavir, HIV protease inhibitors, reduced MMP-9 activity in THP-1 and LX-2 cells, respectively. Plasma MMP-9 activity and expression was higher in HIV-HCV and HIV patients compared with HCV patients and healthy controls. MMP-2 and TIMP-2 levels were similar in all groups. RBV/pegylated IFN-α decreased plasma MMP-9 abundance in HIV-HCV patients. CONCLUSIONS: These data demonstrate that RBV/pegylated IFN-α reduce plasma MMP-9 abundance in vivo and may reduce its activity in vitro through immune cells, such as monocyte/macrophages, rather than hepatic stellate cells. The results of this study indicate that such therapy may mediate tissue remodelling associated with HIV-HCV coinfection through effects on MMP-9.

Nuclear receptor-mediated induction of CYP450 by antiretrovirals: functional consequences of NR1I2 (PXR) polymorphisms and differential prevalence in whites and sub-Saharan Africans.

BACKGROUND: Antiretroviral therapy including HIV protease inhibitors and nonnucleoside reverse transcriptase inhibitors can both inhibit and induce expression of cytochrome P450s, potentially leading to drug interactions. However, information is lacking on the impact of genetic polymorphism on this interaction. METHODS: This study examines the prevalence of 33 polymorphisms in NR1I2 (pregnane X receptor [PXR]), CYP3A4, and CYP2B6 in 1013 white and sub-Saharan African patients with HIV; explores the inductive ability of 16 antiretrovirals on CYP3A4 and CYP2B6 promoter activity through nuclear receptors PXR and constitutive androstane receptor (CAR); and evaluates the influence of naturally occurring PXR genetic variants on antiretroviral activation. RESULTS: Seventeen polymorphisms were present at different frequencies between the two ethnicities. Darunavir, fosamprenavir, lopinavir, nelfinavir, tipranavir, efavirenz, and abacavir increased CYP3A4 and/or CYP2B6 promoter activity, some through constitutive androstane receptor but mainly through PXR. Addition of low-dose ritonavir enhanced levels of CYP promoter activity for several protease inhibitors. Some PXR variants displayed lower fosamprenavir- and lopinavir-induced CYP3A4 promoter activity than the PXR reference sequence, whereas efavirenz and nelfinavir induction was unchanged. CONCLUSIONS: The presence of NR1I2 polymorphisms can alter the induction of CYP3A4 and CYP2B6 promoter activity, potentially adding to the unpredictable nature of antiretroviral drug interactions. These polymorphisms differ in prevalence between whites and sub-Saharan Africans.

Protection of cardiomyocyte function by propofol during simulated ischemia is associated with a direct action to reduce pro-oxidant activity.

To demonstrate a direct protective effect of propofol on myocardial contractile performance during an ischemic episode and investigate underlying mechanisms, isolated adult rat ventricular cardiomyocytes were subjected for 2 h to (i) ischemic medium containing 2-deoxyglucose (20 mM), gassed with 100% N(2) at pH 6.4, (ii) normal medium with 95% O(2)/5% CO(2) at pH 7.4 or (iii) normal medium with addition of H(2)O(2) (50 microM). Propofol under normal conditions decreased the peak amplitude of electrically stimulated contraction of cardiomyocytes from a basal value of 6.5+/-0.37 microm to a maximum attenuation ( approximately 37%) at 0.44 to 56 microM. Under ischemic conditions, the contraction amplitude at baseline was 2.8+/-0.34 microm, but propofol, despite having a cardiodepressant effect per se, stimulated contraction, such that at >or=0.44 microM, normal and ischemic values in the presence of propofol were similar. Comparably, pro-oxidant (H(2)O(2))-induced attenuation of cell shortening was reversed by propofol (0.5 microM) to the level of contractile activity produced by the anaesthetic alone. The protective effect against ischemia-induced injury was not reflected in an improved ATP/ADP ratio nor was it mediated through diltiazem-sensitive L-type Ca(2+) channels. Propofol (0.5 microM) did, however, attenuate the ischemia- and H(2)O(2)-induced increases in the membrane lipid hydroperoxides, MDA (by 83% and 30%) and 4-HNE (by 47% and 69%). It is concluded that propofol, at clinically relevant concentrations, can counteract the effects of increased production of free radical compounds by cardiomyocytes subjected to oxidant stress and improve contractile performance.

Intracellular accumulation of nelfinavir and its relationship to P-glycoprotein expression and function in HIV-infected patients.

OBJECTIVE: To compare plasma and intracellular nelfinavir pharmacokinetics, and determine their relationship to P-glycoprotein (P-gp) expression and function in lymphocytes of HIV-infected patients. METHODS: A pharmacokinetic study of 12 patients receiving nelfinavir plus dual nucleoside analogue therapy. Blood samples were taken at intervals to 12 h. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation, and nelfinavir extracted from cells in the presence of 60% methanol and evaporated to dryness. Both plasma and intracellular nelfinavir samples were assayed by high performance liquid chromatography linked to mass spectrometry. P-gp expression and function were measured by flow cytometric analysis. Data were analysed by non-compartmental analysis using WinNonLin pharmacokinetic software. RESULTS: The mean intracellular nelfinavir AUC(0-12) (mean +/-SE) was about ninefold higher than that of plasma (264,200 +/- 63,420 vs 29,250 +/- 6629 ng/ml/h; P<0.001, and intracellular Cmin and C0 values for nelfinavir were five- to sixfold higher than that of plasma (Cmin: 5712 +/- 2156 vs 1062 +/- 357 ng/ml; C0: 15,860 +/- 3662 vs 2553 +/- 539 ng/ml; P<0.0005). The intracellular nelfinavir Cmax was 15-fold higher than plasma (59,420 +/- 13,940 vs 3986 +/- 822 ng/ml; P<0.0005). There were no differences between plasma and intracellular values for Tmax, elimination half-life or mean residence time. In patients chronically treated with nelfinavir mean P-gp expression was 8.85 +/- 1.3 MFI, there was no correlation between P-gp expression and either intracellular AUC(0-12) (r=-0.35; P=0.29) or intracellular C0 values. There was a correlation between intracellular nelfinavir concentrations and P-gp function at baseline (r=0.59; P<0.05). Basal P-gp-mediated rhodamine efflux was 61.0 +/- 4.2%. In the presence of ritonavir, cellular rhodamine efflux decreased to 25.6 +/- 5.5% (P=0.001), representing an additional reversible efflux potential of 56.1 +/- 9.78%. There was a strong correlation between plasma and intracellular AUC(0-12) for nelfinavir (r=0.75; P=0.011). CONCLUSIONS: Nelfinavir undergoes significant intracellular accumulation within the PBMCs of HIV-infected patients, which may be in part related to its moderate ability to inhibit P-gp-mediated drug efflux. The addition of ritonavir further reduced P-gp function. Intracellular accumulation of nelfinavir correlated with P-gp function but not P-gp expression, suggesting pump activity is substrate concentration-dependant. There was a significant correlation between plasma and intracellular nelfinavir concentrations, suggesting one is a good surrogate marker of the other.

Intracellular indinavir pharmacokinetics in HIV-infected patients: comparison with plasma pharmacokinetics.

OBJECTIVE: To determine intracellular concentrations of indinavir (IDV) and investigate the relationship between plasma and intracellular IDV pharmacokinetics in HIV-infected patients. METHODS: A pharmacokinetic study of 10 patients receiving IDV plus dual nucleoside analogue therapy. Peripheral blood mononuclear cells were isolated by density gradient centrifugation and cell counts estimated. IDV was extracted from cells in the presence of 60% methanol and evaporated to dryness. Both plasma and intracellular IDV samples were assayed by high performance liquid chromatography linked to mass spectrometry. Data were subjected to non-compartmental pharmacokinetic analysis. RESULTS: The mean intracellular IDV area under the curve over 8 h (AUC0-8) was lower than the plasma AUC0-8 (7574 +/- 1003 vs 25060 +/- 4171 ng/ml/h; P<0.004). However, both the elimination half-life (t1/2) and the mean residence time (MRT) of IDV intracellularly were prolonged compared with plasma (t1/2: 2.0 +/- 0.3 vs 1.2 +/- 0.09 h; MRT: 3.6 +/- 0.6 vs 2.1 +/- 0.1 h; P<0.05). All patients were responsive to therapy at the time of the study, as assessed by HIV plasma RNA levels. Individual plasma versus intracellular time course results suggest that, due to the prolonged intracellular half-life, some patients may achieve acceptable intracellular IDV concentrations despite sub-therapeutic plasma levels. Similarly, potentially inadequate intracellular concentrations may occur despite therapeutic plasma concentrations. CONCLUSIONS: There is no significant intracellular accumulation of IDV within the lymphocytes of HIV-1-infected patients relative to plasma. However, intracellular concentrations are compatible with reported IDV-free drug concentrations in plasma. The intracellular elimination half-life and mean residence time of IDV are significantly prolonged compared with plasma. This may in part explain why certain patients maintain adequate viral suppression despite sub-therapeutic plasma IDV levels.

Neuropeptide Y Y(1) receptor regulates protein turnover and constitutive gene expression in hypertrophying cardiomyocytes.

Increased levels of neuropeptide Y correlate with severity of left ventricular hypertrophy in vivo. At cardiomyocyte level, hypertrophy is characterised by increased mass and altered phenotype. The aims were to determine the contributions of increased synthesis and reduced degradation of protein to neuropeptide Y-mediated increase in mass, assess effects on gene expression, and characterise neuropeptide Y Y receptor subtype involvement. Neuropeptide Y (10 nM) increased protein mass of adult rat ventricular cardiomyocytes maintained in culture (24 h) (16%>basal) and de novo protein synthesis (incorporation of [(14)C]phenylalanine) (18%>basal). Neuropeptide Y (100 nM) prevented degradation of existing protein at 8 h. Actinomycin D (5 microM) attenuated increases in protein mass to neuropeptide Y (< or = 1 nM) but not to neuropeptide Y (10 nM). [Leu(31), Pro(34)]neuropeptide Y (10 nM), an agonist at neuropeptide Y Y(1) receptors, increased protein mass (25%>basal) but did not stimulate protein synthesis. Neuropeptide Y-(3-36) (10 nM), an agonist at neuropeptide Y Y(2) receptors, increased protein mass (29%>basal) and increased protein synthesis (13%>basal), respectively. Actinomycin D (5 microM) abolished the increase in protein mass elicited by neuropeptide Y-(3-36) but not that by [Leu(31), Pro(34)]neuropeptide Y. BIBP3226 [(R)-N2-(diphenylacetyl)-N-(4-hydroxyphenylmethyl)-D-arginine amide] (1 microM), a neuropeptide Y Y(1) receptor subtype-selective antagonist, and T(4) [neuropeptide Y-(33-36)](4), a neuropeptide Y Y(2) receptor subtype-selective antagonist, attenuated the increase in protein mass to 100 nM neuropeptide Y by 68% and 59%, respectively. Neuropeptide Y increased expression of the constitutive gene, myosin light chain-2 (MLC-2), maximally at 12 h (4.7-fold>basal) but did not induce (t< or = 36 h) expression of foetal genes (atrial natriuretic peptide (ANP), skeletal-alpha-actin and myosin heavy chain-beta). This increase was attenuated by 86% and 51%, respectively, by BIBP3226 (1 microM) and T(4) [neuropeptide Y-(33-36)](4) (100 nM). [Leu(31), Pro(34)]neuropeptide Y (100 nM) (2.4-fold>basal) and peptide YY-(3-36) (100 nM) (2.3 fold>basal) increased expression of MLC-2 mRNA at 12 h. In conclusion, initiation of cardiomyocyte hypertrophy by neuropeptide Y requires activation of both neuropeptide Y Y(1) and neuropeptide Y Y(2) receptors and is associated with enhanced synthesis and attenuated degradation of protein together with increased expression of constitutive genes but not reinduction of foetal genes.